Tendinopathies are ubiquitous and age-related. The natural history of tendinosis includes age-related tissue degeneration of the tendon mid-substance, or of the enthesis ? the tendon-bone interface ? often resulting in acute pain, loss of function and chronic disability. The primary reason for poor outcomes after surgical repair is failure of the endogenous cells to recreate fibrocartilage at the tendon-bone interface. Our prior studies of aged and young human tendon have revealed tenocytes develop a fibrochondrocyte and mineralized fibrochondrocyte phenotype depending on relative intracellular levels of Rac1 and RhoA GTPase activity, which are determined by the cell microenvironment. We have characterized this effect of RhoA/Rac1 GTPase activity on the human tenocyte phenotype, and have discovered an essential role of combined Rac1 in-activity and RhoA over-activity in development of the fibrochondrocyte phenotype. The goal of this proposal is to create a fibrocartilage construct fit for eventual clinical translation. First we will create a human tenocyte-engrafted electrospun nanofiber scaffold and culture it under conditions of hypoxia or normoxia, under tension or compression in a bioreactor. Biochemical, immunohistochemical and kinematic assays will be used to evaluate time and oxygen-dependent changes in human tenocyte phenotype on the scaffolds, via expression of selected markers, assessment of tissue organization and mechanical strength. Human tenocytes will then be treated with RhoA/Rac1 agents to produce Rac-1 in-activity and RhoA over- activity and cultured as above. The final objective of this proposal is to create an in vivo rabbit model for enthesis regeneration. First we will perform proof-of-concept studies: enthesis defects will be created in the Achilles tendon of rabbits and observed for healing at week 6 and 12 after defect creation, harvested and analyzed as above. Using the same enthesis defect model, tenocyte-engrafted scaffolds with stable Rac1 in-activity and RhoA over-activity will then be used to repair the enthesis defects, harvested at week 6 and 12, and analyzed as described above to evaluate maintenance of the enthesis phenotype as well as construct integrity during physiologic loading in vivo. Our aging patient population is in dire need of an innovative, consistent therapy for tendinosis. Our proposed interdisciplinary approach using cell biology, biochemical, tissue engineering and translational techniques will create a novel enthesis construct, fit to undergo clinical testing, with the goal of decreasing pain and restoring function in the elderly.